VIP and muscarinic synergistic mucin secretion by salivary mucous cells is mediated by enhanced PKC activity via VIP-induced release of an intracellular Ca2+ pool.

Mucin secretion by salivary mucous glands is mediated predominantly by parasympathetic acetylcholine activation of cholinergic muscarinic receptors via increased intracellular free calcium ([Ca2+]i) and activation of conventional protein kinase C isozymes (cPKC). However, the parasympathetic co-neurotransmitter, vasoactive intestinal peptide (VIP), also initiates secretion, but to a lesser extent. In the present study, cross talk between VIP- and muscarinic-induced mucin secretion was investigated using isolated rat sublingual tubuloacini. VIP-induced secretion is mediated by cAMP-activated protein kinase A (PKA), independently of increased [Ca2+]i. Synergistic secretion between VIP and the muscarinic agonist, carbachol, was demonstrated but only with submaximal carbachol. Carbachol has no effect on cAMP ± VIP. Instead, PKA activated by VIP releases Ca2+ from an intracellular pool maintained by the sarco/endoplasmic reticulum Ca2+-ATPase pump. Calcium release was independent of phospholipase C activity. The resultant sustained [Ca2+]i increase is additive to submaximal, but not maximal carbachol-induced [Ca2+]i. Synergistic mucin secretion was mimicked by VIP plus either phorbol 12-myristate 13-acetate or 0.01 µM thapsigargin, and blocked by the PKC inhibitor, Gö6976. VIP-induced Ca2+ release also promoted store-operated Ca2+ entry. Synergism is therefore driven by VIP-mediated [Ca2+]i augmenting cPKC activity to enhance muscarinic mucin secretion. Additional data suggest ryanodine receptors control VIP/PKA-mediated Ca2+ release from a Ca2+ pool also responsive to maximal carbachol. A working model of muscarinic and VIP control of mucous cell exocrine secretion is presented. Results are discussed in relation to synergistic mechanisms in other secretory cells, and the physiological and therapeutic significance of VIP/muscarinic synergism controlling salivary mucous cell exocrine secretion.

The impact of different hair-removal behaviours on the biophysical and biochemical characteristics of female axillary skin.

OBJECTIVE: The impact of hair removal on the biophysical and biochemical characteristics of human axillary skin is not fully understood. This study investigated the effect of different hair-removal techniques on biophysical parameters and the concentrations of key inflammatory biomarkers in the axillae of female Thai subjects. Axillary hair was removed by shaving, plucking or waxing. METHODS: Following a 2-week washout phase without hair removal, subjects underwent visual assessment for erythema and skin dryness in one (randomized) axilla, then, hair was removed from the axilla by shaving, plucking or waxing according to each subject's established habit. Erythema and dryness were assessed again 30 min after hair removal, and buffer scrubs collected from depilated and non-depilated axillae and analysed for inflammatory cytokines; after a further 48 h, erythema, dryness and post-inflammatory hyperpigmentation (PIHP) were assessed in the depilated axilla. Biophysical assessments (skin hydration, barrier integrity, elasticity and roughness) were made in depilated and non-depilated axillae. RESULTS: All three hair-removal techniques induced an increase in axillary erythema and skin dryness. Shaving was associated with significantly less erythema (P < 0.01), but significantly greater skin dryness (P < 0.05) versus the other techniques 30 min after hair removal. There were no between-technique differences in PIHP or biophysical parameters. Interleukins IL-1α and IL-1RA concentrations increased, and IL-8 concentration decreased following hair removal by each technique. CONCLUSION: This is the first study to identify the principal cytokines associated with the inflammatory process triggered by axillary hair removal. A single hair-removal treatment did not appear to induce PIHP or further biophysical changes to the skin.

OBJECTIF: L'impact de l'épilation sur les caractéristiques biophysiques et biochimiques de la peau axillaire humaine n'est pas entièrement compris. Cette étude a examiné l'effet de différentes techniques d'épilation sur les paramètres biophysiques et les concentrations de biomarqueurs inflammatoires clés dans les aisselles de sujets thaïlandais de sexe féminin. Les aisselles ont été épilées par rasage, à la pince ou à la cire. MÉTHODES: Après une phase de sevrage de 2 semaines sans épilation, les sujets ont subi une évaluation visuelle de l'érythème et de la sécheresse cutanée dans une aisselle (randomisé), puis l'aisselle a été épilée par rasage, à la pince ou à la cire selon l'habitude établie de chaque sujet. L'érythème et la sécheresse ont été évalués à nouveau 30 minutes après l'épilation, et des frottis tampons ont été prélevés dans les aisselles épilées et non épilées et analysés pour détecter les cytokines inflammatoires; puis, après 48 heures, l'érythème, la sécheresse et l'hyperpigmentation post-inflammatoire (PIHP) ont été évalués dans les aisselles épilées. Des évaluations biophysiques (hydratation cutanée, intégrité de la barrière cutanée, élasticité et rugosité de la peau) ont été réalisées sur les aisselles épilées et non épilées. RÉSULTATS: Les trois techniques d'épilation ont entraîné une accentuation de l'érythème axillaire et de la sécheresse de la peau. Le rasage a été associé à un érythème nettement moins important (P < 0,01), mais à une sécheresse cutanée nettement plus importante (P < 0,05) par rapport aux autres techniques 30 min après l'épilation. Aucune différence entre les techniques n'a été observée en ce qui concerne le PIHP ou les paramètres biophysiques. Les concentrations en interleukines IL-1α IL-1RA ont augmenté, et la concentration en IL-8 a diminué après l'épilation par chaque technique. CONCLUSION: Cette étude est la première à identifier les cytokines principales associées au processus inflammatoire déclenché par l'épilation des aisselles. Une seule épilation n'a pas semblé entraîner de PIHP ou d'autres modifications biophysiques de la peau.

Melt-rich channel observed at the lithosphere-asthenosphere boundary.

The lithosphere-asthenosphere boundary (LAB) separates rigid oceanic plates from the underlying warm ductile asthenosphere. Although a viscosity decrease beneath this boundary is essential for plate tectonics, a consensus on its origin remains elusive. Seismic studies identify a prominent velocity discontinuity at depths thought to coincide with the LAB but disagree on its cause, generally invoking either partial melting or a mantle dehydration boundary as explanations. Here we use sea-floor magnetotelluric data to image the electrical conductivity of the LAB beneath the edge of the Cocos plate at the Middle America trench offshore of Nicaragua. Underneath the resistive oceanic lithosphere, the magnetotelluric data reveal a high-conductivity layer confined to depths of 45 to 70 kilometres. Because partial melts are stable at these depths in a warm damp mantle, we interpret the conductor to be a partially molten layer capped by an impermeable frozen lid that is the base of the lithosphere. A conductivity anisotropy parallel to plate motion indicates that this melt has been sheared into flow-aligned tube-like structures. We infer that the LAB beneath young plates consists of a thin, partially molten, channel of low viscosity that acts to decouple the overlying brittle lithosphere from the deeper convecting mantle. Because this boundary layer has the potential to behave as a lubricant to plate motion, its proximity to the trench may have implications for subduction dynamics.

Axillary skin: biology and care.

In skin care, the axilla is a biologically unique site requiring specialized attention and care. This area of skin is often subject to hair removal techniques, such as shaving and plucking. These procedures damage the skin leading to erythema and dryness in the short term, and in some cases, post-inflammatory hyperpigmentation (PIHP) in the long term. This study will (i) briefly review the biology and unique properties of axillary skin, and (ii) describe the characteristics of the irritation and damage induced by contemporary skin care habits and resolution of these responses by the use of efficacious skin moisturizing technology. With respect to the latter, we propose that there are five groups of compounds, defined according to their mechanism of action, which are particularly relevant to the care of damaged axillary skin.

Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens.

UV-induced mutations in the p53 tumor suppressor gene play an essential role in skin cancer development. We report here that such mutations can be detected in UV-irradiated mouse skin months before the gross appearance of skin tumors. Application of SPF-15 sunscreens to mouse skin before each UV irradiation nearly abolished the frequency of p53 mutations. These results indicate that p53 mutation is an early event in UV skin carcinogenesis and that inhibition of this event may serve as an early end point for assessing protective measures against skin cancer development.

Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocyte subpopulations block the mixed leukocyte reaction in man.

Two major subsets of human T lymphocytes that are functionally analogous to the mouse Lyt-2+ and Lyt-2- subsets have been defined by their expression of two thymus-dependent membrane antigens, Leu-2 and Leu-3. Leu-2+,3- cells have suppressor/cytotoxic functions and Leu-2-,3+ cells have helper functions. These studies were designed to determine the effects of adding IgG1 monoclonal anti-Leu-2 and anti-Leu-3 antibodies to the mixed leukocyte reaction (MLR). At high concentrations, each antibody partially inhibited the proliferative response of unseparated T cells and abolished the response of the isolated subset having the appropriate phenotype. An IgG1 monoclonal antibody to HLA-A2 and an IgG2a antibody to Leu-1, a pan-T antigen, failed to inhibited the MLR. These results suggest that the Leu-2 and Leu-3 antigens may have a direct role in the mechanism whereby T cells recognize and respond to alloantigen.

Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells.

Aggregation of the high affinity receptor for immunoglobulin E (Fc epsilon RI) on mast cells results in rapid tyrosine phosphorylation and activation of Syk, a cytoplasmic protein tyrosine kinase. To examine the role of Syk in the Fc epsilon RI signaling pathway, we identified a variant of RBL-2H3 cells that has no detectable Syk by immunoblotting and by in vitro kinase reactions. In these Syk-deficient TB1A2 cells, aggregation of Fc epsilon RI induced no histamine release and no detectable increase in total cellular protein tyrosine phosphorylation. However, stimulation of these cells with the calcium ionophore did induce degranulation. Fc epsilon RI aggregation induced tyrosine phosphorylation of the beta and gamma subunits of the receptor, but no increase in the tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2 and no detectable increase in intracellular free Ca2+ concentration. By transfection, cloned lines were established with stable expression of Syk. In these reconstituted cells, Fc epsilon RI aggregation induced tyrosine phosphorylation of phospholipase C-gamma 1 and phospholipase C-gamma 2, an increase in intracellular free Ca2+ and histamine release. These results demonstrate that Syk plays a critical role in the early Fc epsilon RI-mediated signaling events. It further demonstrates that Syk activation occurs downstream of receptor phosphorylation, but upstream of most of the Fc epsilon RI-mediated protein tyrosine phosphorylations.

Antibody-dependent cellular cytotoxicity by allosensitized human T cells.

Peripheral human T cells, isolated by sheep erythrocyte-rosette formation and density centrifugation, were highly cytotoxic to both Ab-coated autologous lymphocytes and antibody (Ab)-coated chicken erythrocytes when stimulated in mixed lymphocyte culture, but were not lytic when freshly purified, or when unstimulated in 6-day culture. Allosensitized T cells were shown to effect this activity by a specific effector-target cell interaction dependent on Ab, as indicated by: (a) induction of killing by Ab to target cells not lysed in the absence of Ab. (b) inhibition of Ab-dependent killing by aggregated Ig. The mechanism by which allosensitized T cells effect antibody-dependent cellular cytotoxicity is discussed.

Identification of a p69,71 complex expressed on human T cells sharing determinants with B-type chronic lymphatic leukemic cells.

In the course of generating monoclonal antibodies to human thymus-dependent differentiation antigens, we were able to define specificities shared by T cells and by cells from patients with chronic lymphatic leukemia that were not detectable on normal B cells. In particular, one of these antibodies was reactive by indirect immunofluorescence with greater than 95% of the thymocytes and 80--95% of nonadherent sheep erythrocyte-rosetting peripheral blood lymphocytes (PBL), but was unreactive with normal B cells or cell lines derived from PBL by Epstein-Barr virus transformation. However, the leukemic cells from 11 of 14 patients with B-type chronic lymphatic leukemia were found to express detectable concentrations of this surface determinant. The target antigen recognized by this monoclonal antibody was shown by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a p69,71 complex. Our findings suggest a possible relationship between this antigen and the previously described GIX system in the mouse.

Detection, isolation, and functional characterization of two human T-cell subclasses bearing unique differentiation antigens.

A heterologous antihuman T-cell serum (anti-TH1), raised against purified peripheral T cells, and absorbed with an autologous Ig+ line, was shown to bind specifically to T- but not to B-lymphoid cells by both a complement-dependent cytotoxic assay and indirect immunofluorescence. Whereas 90% fetal thymocytes and thymocytes were killed by anti-TH1 and complement, a consistently restricted population (50-60%) of peripheral T cells from several normal donors were lysed, indicating that anti-TH1 is directed against one or more thymus-specific antigens which are lost or reduced on a subpopulation of human T cells in the periphery. Functional analysis of the unreactive (TH1-) and reactive (TH1+) T-cell subclasses demonstrated that TH1- cells mounted a good proliferative response to a battery of specific soluble antigens (mumps, PPD, tetanus toxoid) but neither responded in MLC, nor elaborated LMF in response to tetanus toxoid. In contrast TH1+ cells proliferated in MLC and elaborated LMF but did not respond by 3H-incorporation to soluble antigens. The relevance of these findings to human T-cell functions in vivo and to previously described functional subclasses of murine T cells is discussed.

Evolutionary conservation of surface molecules that distinguish T lymphocyte helper/inducer and cytotoxic/suppressor subpopulations in mouse and man.

We describe the biochemical properties and cell surface distributions of three human T cell antigens (Leu-1, Leu-2a, and Leu-2b) which we postulate to be the homologues of the Lyt-1, Lyt-2, and Lyt-3 antigens that distinguish functional T cell subsets in the mouse. Leu-l, like Lyt-1, is on all thymocytes and peripheral T cells and is present in greater amounts on the helper/inducer subset than on the cytotoxic/suppressor subset. Both antigens increase in parallel fashion during T cell maturation in the thymus and each antigen is carried on a single 67,000-molecular weight (relative) (M(r)) polypeptide chain. Surprisingly, Leu-1 and Lyt-1 each are also expressed in readily detectable amounts on some B celI Ieukemias but not detectably so on normal B cells. Leu-2a and Leu-2b are antigens found only on suppressor/cytotoxic cells in the human and are very similar to the murine Lyt-2 and Lyt-3 antigens. In both species, the two antigens are on the same disulfide- linked multimeric molecules. Disulfide-bond reduction in both species yields subunits of similar size and charge. Lyt-3 and Leu-2b are extremely sensitive to trypsin digestion on viable cells whereas Lyt-2 and Leu-2a are much less so. A different membrane antigen, Leu-3, is an exclusive marker of the helper/inducer subset in man. No mouse homologue for this 55,000-M(r) protein is known. The maintenance of the homologous molecules on functionally distinct T cell subpopulations in two evolutionarily distant species suggests that the Lyt and Leu antigens perform essential functions for the cells on which they are found.

Peripheral human T cells sensitized in mixed leukocyte culture synthesize and express Ia-like antigens.

We have studied the modulation of Ia-like antigens on the surface membrane of human T cells responding in a one-way mixed leukocyte culture. A heterologous antiserum, (anti-p23,30), which is specific to HLA-D-related antigens and which is unreactive with normal peripheral T cells or thymocytes, was found to bind significantly to all T cells transformed in mixed leukocyte culture (MLC) as determined by indirect immunofluorescence on a fluorescence-activated cell sorter 1. Furthermore, cytotoxic T cells responsible for cell-mediated lympholysis were shown to react with anti-p23,30, whereas their unactivated progenitors did not. Immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis of a radioactive 29,000 and 34,000 dalton complex from MLC-primed T cells labeled with [35S]methionine indicated that allosensitized T cells synthesized these HLA-D-related antigens.

Certification of standard reference materials containing bitter orange.

A suite of three dietary supplement standard reference materials (SRMs) containing bitter orange has been developed, and the levels of five alkaloids and caffeine have been measured by multiple analytical methods. Synephrine, octopamine, tyramine, N-methyltyramine, hordenine, total alkaloids, and caffeine were determined by as many as six analytical methods, with measurements performed at the National Institute of Standards and Technology and at two collaborating laboratories. The methods offer substantial independence, with two types of extractions, two separation methods, and four detection methods. Excellent agreement was obtained among the measurements, with data reproducibility for most methods and analytes better than 5% relative standard deviation. The bitter-orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods.

Survivorship of the St Georg Sled medial unicompartmental knee replacement beyond ten years.

There have been several reports of good survivorship and excellent function at ten years with fixed-bearing unicompartmental knee replacement. However, little is known about survival beyond ten years. From the Bristol database of over 4000 knee replacements, we identified 203 St Georg Sled unicompartmental knee replacements (174 patients) which had already survived ten years. The mean age of the patients at surgery was 67.1 years (35.7 to 85) with 67 (38.5%) being under 65 years at the time of surgery. They were reviewed at a mean of 14.8 years (10 to 29.4) from surgery to determine survivorship and function. There were 99 knees followed up for 15 years, 21 for 20 years and four for 25 years. The remainder failed, were withdrawn, or the patient had died. In 58 patients (69 knees) the implant was in situ at the time of death. Revision was undertaken in 16 knees (7.9%) at a mean of 13 years (10.2 to 21.6) after operation. In seven knees (3.4%) this was for progression of arthritis, in three (1.5%) for wear of polyethylene, in four (2%) for tibial loosening, in two (1%) for fracture of the femoral component and in two (1%) for infection. Two knees (1%) were revised for more than one reason. The mean Bristol knee score of the surviving knees fell from 86 (34 to 100) to 79 (42 to 100) during the second decade. Survivorship to 20 years was 85.9% (95% CI 82.9% to 88.9%) and at 25 years was 80% (95% CI 70.2% to 89.8%). Satisfactory survival of a fixed-bearing unicompartmental knee replacement can be achieved into the second decade and beyond.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis.

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC-/- mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC-/- skin tumor analysed displayed C-->T or CC-->TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC-/- and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC-/- mouse skin after 3 - 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC-/- mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC-/- mice.

Intracellular calcium transients in suctorian protozoa (Trichophrya spp.): correlation with spontaneous tentacle contractions.

The luminescent photoprotein aequorin was used to measure intracellular free Ca2+ in three species of suctorian protozoon, Trichophrya riederi, Trichophrya collini and Trichophrya rotunda. Resting [Ca2+]i ranged from about 75-110 nM, and was unaffected by a change in temperature of the perfusate. Spontaneous Ca2+ transients were observed in all three species, with peak amplitudes ranging from 100-600 nM. In T. riederi and T. rotunda, three categories of transient (small, intermediate, large) were recorded; T. collini displayed only small transients. In both T. riederi and T. collini, raising the temperature from 5 degrees to 26 degrees C led to an increase in the frequency of transients. Furthermore, in T. riederi, large transients occurred only at the higher temperature. The frequency of spontaneous contractions of the tentacles of T. riederi was also temperature-dependent. Increasing the temperature over the range 5-26 degrees C led to a concomitant increase in contraction frequency and a decrease in mean tentacle length. The possible mechanisms of spontaneous Ca2+ transient generation and their role in the control of contraction are discussed.

An evaluation of population pharmacokinetics in therapeutic trials. Part II. Detection of a drug-drug interaction.

The use of observational data, collected during the routine clinical care of patients, has been advocated as a means to obtain clinically relevant information regarding the pharmacokinetic parameters of drugs. However, the validity of this approach and its proper role in new drug development is unclear. This study was performed to evaluate the ability of three approaches to estimate population pharmacokinetic parameters: the traditional approach, mixed-effect modeling, and a simple pharmacokinetic screen. The evaluation was performed with data collected during a multicenter, open-label study evaluating the efficacy, safety, and pharmacokinetics of imipramine and alprazolam in combination. The traditional pharmacokinetic study demonstrated a 20% decrease in the clearance of imipramine in the presence of 4 mg/day alprazolam. Mixed-effect modeling extends these findings by suggesting that the interaction is dependent on the simultaneous concentration of alprazolam, a finding that was not possible under the study design typically used for traditional pharmacokinetic studies. Although the simple screen suggests the presence of the drug-drug interaction, limited information regarding pharmacokinetic parameters is available and those parameters that can be estimated are biased.

Graphic analysis of superoxide secretion by phagocytic leukocytes at different cell concentrations.

Superoxide secretion by monocytes, macrophages and neutrophils becomes less efficient in a non-linear manner as the cell concentration is increased. This relation holds for cells in suspension or attached to a substrate, elicited into the peritoneum with various agents or from the peripheral circulation, acutely stimulated with particulate or soluble agents, and in experimental animals and in man. The significant observation of this study is that in all these systems, of data from our own studies as well as from the literature, plots of the logarithm of superoxide secreted per cell per unit of time versus the logarithm of the cell concentration were linear with correlation coefficients better than 0.98. The common practice of comparing data on superoxide secretion from experiments performed at different cell concentrations is clearly unsatisfactory. It is suggested that superoxide secretion be expressed in terms of both the slope of the log log plot and the secretion rate at a given cell concentration.

Characterization of B-cell type chronic lymphocytic leukemia cells by surface markers and a monoclonal antibody.

This study was carried out to determine the reactivity of the Leu-1 mouse monoclonal antibody with B-chronic lymphocytic leukemia and other B-cell leukemias. This antibody has been previously reported to recognize a surface antigen expressed by almost all human thymocytes and peripheral T cells. It was also detected on surface immunoglobulin-bearing cells of most patients with chronic lymphocytic leukemia but was not detectable in normal B-cells and B-cell lines. In the present series, the neoplastic lymphocytes in 33 cases of B-chronic lymphocytic leukemia expressed surface immunoglobulin, Ia antigen, and receptors for Fc, C3, and mouse erythrocytes. All but one case expressed the Leu-1 antigen as detected by indirect immunofluorescence using flow cytometry. In three other cases, the leukemic cells in the peripheral blood reacted with anti-Leu-1, whereas the bone marrow lymphocytes did not. Moreover, quantitative differences in the surface density of Leu-1 were apparent by flow cytometry. The peripheral blood and bone marrow lymphocytes in other B-cell leukemias, including 15 cases of leukemic B-cell lymphomas and three cases of hairy-cell leukemia, failed to stain positively with the anti-Leu-1 antibody. The recognition of the Leu-1 antigen adds to the phenotypic characterization of b-chronic lymphocytic leukemia and may contribute to a better understanding of the pathogenesis of the disease and its expression in different tissues.

Identification with the monoclonal antibody 26.163 of a determinant shared by the beta chains of the gene products of the HLA-DR, DQ, and DP loci.

Immunochemical studies (sequential immunoprecipitation followed by SDS-PAGE and two-dimensional gel electrophoresis) have shown that the monoclonal antibody (MoAb) 26.163 reacts with HLA-DR, DQ, and DP antigens. Testing with isolated alpha and beta chains of HLA class II antigens and immunoblot analysis also demonstrated that the determinant defined by the MoAb 26.163 is localized on beta chains and does not require their association with alpha chains for its expression. The MoAb 26.163 appears to be the first example of a monoclonal antibody with specificity for the gene products of HLA-DR, DQ, and DP loci.